INTERNATIONAL JOURNAL OF 58: 10, 2021

Current molecular and clinical insights into uveal (Review)

MATTEO FALLICO1*, GIUSEPPINA RACITI2*, ANTONIO LONGO1, MICHELE REIBALDI3, VINCENZA BONFIGLIO4, ANDREA RUSSO1, ROSARIO CALTABIANO5, GIUSEPPE GATTUSO6, LUCA FALZONE7 and TERESIO AVITABILE1

1Department of Ophthalmology, University of Catania, I‑95123 Catania; 2Department of Drug Sciences, Section of Biochemistry, University of Catania, I‑95125 Catania; 3Department of Surgical Sciences, Eye Clinic Section, University of Turin, I‑10122 Turin; 4Department of Experimental Biomedicine and Clinical Neuroscience, Ophthalmology Section, University of Palermo, I‑90127 Palermo; 5Department ‘G.F. Ingrassia’, Section of Anatomic Pathology, and 6Department of Biomedical and Biotechnological Sciences, University of Catania, I‑95123 Catania; 7Epidemiology Unit, IRCCS Istituto Nazionale Tumori ‘Fondazione G. Pascale’, I‑80131 Naples, Italy

Received October 27, 2020; Accepted November 30, 2020

DOI: 10.3892/ijo.2021.5190

Abstract. (UM) represents the most aggressiveness of the condition. Novel molecular studies prominent primary eye cancer in adults. With an incidence have allowed for a better understanding of the genetic of approximately 5 cases per million individuals annually and epigenetic mechanisms involved in UM biological in the United States, UM could be considered a relatively activity, which differs compared to skin . The rare cancer. The 90‑95% of UM cases arise from the most commonly mutated genes are GNAQ, GNA11 and . Diagnosis is based mainly on a clinical examina‑ BAP1. Research in this field could help to identify effec‑ tion and ancillary tests, with ocular ultrasonography being tive diagnostic and prognostic biomarkers, as well as novel of greatest value. Differential diagnosis can prove chal‑ therapeutic targets. lenging in the case of indeterminate choroidal lesions and, sometimes, monitoring for documented growth may be the proper approach. Fine needle aspiration biopsy tends to be Contents performed with a prognostic purpose, often in combination with radiotherapy. Gene expression profiling has allowed for 1. Introduction the grading of UMs into two classes, which feature different 2. Epidemiology metastatic risks. Patients with UM require a specialized 3. Risk factors multidisciplinary management. Primary tumor treatment 4. Clinical characteristics can be either enucleation or globe preserving. Usually, 5. Diagnosis enucleation is reserved for larger tumors, while radiotherapy 6. Staging is preferred for small/medium melanomas. The prognosis is 7. Prognosis unfavorable due to the high mortality rate and high tendency 8. Primary tumor treatment to metastasize. Following the development of metastatic 9. Surveillance and metastatic disease treatment disease, the mortality rate increases to 80% within one year, 10. Genetic and epigenetic features in uveal melanoma due to both the absence of an effective treatment and the 11. Conclusions

1. Introduction

Correspondence to: Dr Luca Falzone, Epidemiology Unit, IRCCS Uveal melanoma (UM) represents the most prominent primary Istituto Nazionale Tumori ‘Fondazione G. Pascale’, Via Mariano eye cancer in adults. Although it could be considered a rela‑ Semmola 53, I‑80131 Naples, Italy E‑mail: [email protected] tively rare tumor, UM remains a disease of primary interest due to its high mortality rate. *Contributed equally Notable efforts have been made to improve the manage‑ ment of this malignancy. The introduction of globe‑sparing Key words: uveal melanoma, staging, treatment, diagnosis, treatments was the most significant breakthrough of the last prognosis, biomarkers, epigenetics century in this field. However, the overall survival of affected patients has remained unaltered, and there is still no effective treatment for metastatic disease. 2 FALLICO et al: INSIGHTS INTO UVEAL MELANOMA

Recently, increasing attention has focused on the 3. Risk factors molecular mechanisms involved in UM carcinogenesis and progression, which could allow for the identification of valu‑ Several risk factors have been associated with the develop‑ able diagnostic and prognostic biomarkers, as well as novel ment of UM. Host susceptibility variables, such as fair skin therapeutic targets. color, inability to tan and light have been signifi‑ Herein, the available evidence on epidemiological, clinical cantly associated with UM, with a risk ratio of 1.80, 1.64 and and molecular aspects of UM is discussed and reviewed, with 1.75, respectively (19). This association is likely to be related an aim of providing an updated and comprehensive tool, which to the poor amount of in the skin and eyes. It has may be useful for both clinicians and researchers. been assumed that a poor amount of melanin is present in the choroid and retinal pigment epithelium, leading to an 2. Epidemiology increased susceptibility to light and a higher risk of developing UM (19). Oculodermal melanocytosis, also known UM represents 3‑5% of all melanomas (1,2). The largest as , represents a relevant risk factor for developing proportion of cases of UM, approximately 85‑90%, arises UM (20,21). This condition is characterized by an abnormal from the choroid, while the 5‑8% arises from the ciliary congenital hyper‑pigmentation within the V1/V2 trigeminal body and the 3‑5% from the (2,3). The incidence nerve area, and can involve periocular skin, orbit, , sclera of UM in the United States, between 1973 and 2008, and conjunctiva, as well as the palate, meninges and tympanic amounted to 5.1 cases per million annually (4). In Europe, membrane (20,21). Usually this condition is unilateral and, a cancer registry‑based study demonstrated a north‑to‑south is mostly confined to the eye. Oculodermal melanocytosis is decreasing gradient in the incidence of UM, with an inci‑ 35‑fold more common in patients with UM compared to the dence of >8 cases per million in Norway and Denmark healthy population: The incidence rate among the Caucasian compared with approximately 2 cases per million in Spain population is 0.04 vs. 1.4% to 3% in patients with UM (20,21). and Southern Italy (5). Likewise, in Africa and Asia, the A Caucasian patient affected by oculodermal melanocytosis incidence is low, amounting to 0.2‑0.3 cases per million presents a lifetime risk for developing UM equal to 1:400 (22). per year (6). This latitude‑related decreasing trend in the The presence of atypical cutaneous nevi and intraocular incidence of UM has been associated with the protective nevi has been also associated with the development of UM. role of ocular pigmentation, which is higher in southern In particular, the risk of developing UM is 4‑10‑fold higher countries compared to northern ones (5). Similarly, dark in patients affected by atypical cutaneous nevi than in the skin pigmentation may play a role in protecting populations healthy population (23,24). Intraocular nevi, such as iris nevi of African origin: The ratio of UM among populations of and choroidal nevi, are considered risk factors for UM. Iris African origin vs. Caucasian populations ranges from 1:15 nevi have been reported to have a potential risk of malignant to 1:50 (7‑9). A population‑based study investigated the rela‑ transformation, although the rate of this transformation has not tive risk of UM in several racial cohorts, revealing a 5‑fold been clearly established, ranging from 2‑5% (25,26). Predictive higher risk among Hispanic populations and 19‑fold higher factors for an iris nevus to transform into an iris melanoma risk among non‑Hispanic Caucasian populations compared have been summarized in the ABCDEF acronym: A stands populations of African origin (9). for young age; B stands for ; C stands for clock‑hour UM is typically an adult malignancy, affecting older age (inferior location); D stands for diffuse flat shape; E stands for groups (10). The median age at diagnosis has been reported ectropion uveae; F stands for feathery margins (26). Choroidal to be approximately 62 years (2), with an incidence rate that nevus is a common finding in the healthy population, with an tends to increase progressively up to 70 years of age, and incidence rate of approximately 5% in the United States (27). then levels off after 75 years of age (4,5,10,11). The mean age Based on the concept that all melanomas originate from a at diagnosis for UM seems to decrease from 59‑62 years of nevus, the rate of transformation of a choroidal nevus into age in Caucasians, (4,12) to 55 years in Japanese, 51 years in melanoma has been reported as 1:8845, increasing to 1:3664 in Taiwanese and 45 years in Chinese populations (13‑15). UM the older aged cohort (80‑84 years old) (28). Predictive factors is uncommon in children and extremely rare in newborns; for a choroidal melanoma to become malignant are a thickness congenital melanoma is also rare (11,16,17). Shields et al of >2 mm, the presence of subretinal fluid, presence of orange investigated the incidence rate of UM in children and teenagers, pigment, proximity to optic disc, the absence of drusen or halo and demonstrated that 50% of cases were >15 years of age, 35% and ultrasonographic hollowness (29,30). were between 10 and 15 years, 11% between 5 and 10 years, A further relevant risk factor for the development of UM is and only 3% of cases were between 0 and 5 years of age at the the mutation of the onco‑suppressor gene, BRCA1 associated time of diagnosis (17). protein 1 (BAP1) (10,31). BAP1 is located on . The incidence of UM appears to be related to sex as The mutation of this gene has been associated with a hereditary well (2,10). Population‑based studies have demonstrated a cancer syndrome. Tumors, such as malignant , higher age‑adjusted incidence in the male sex compared basal cell carcinoma, cutaneous melanomas, UMs and renal to females, with a 20‑30% greater rate in males (1,4). An cell carcinoma, can be developed following either the somatic Australian population‑based study found this difference to be or germline mutation of BAP1 (31). In the case of germline more prominent in the population which was ≥65 years of age, mutation, the tumors seem to be less aggressive than those whilst there was no significant difference in sex as regards the without this mutation (31). BAP1 has been found mutated in incidence of UM when considering the population <65 years up to 47% of UM cases (32). Of note, patients affected by of age (18). UM present a higher risk compared to the general population INTERNATIONAL JOURNAL OF ONCOLOGY 58: 10, 2021 3

(approximately ≥11%) of a developing a secondary cancer, at the time of diagnosis was relatively large (42). However, including renal cell carcinoma and cutaneous melanoma, the same study also included 7,256 choroidal melanomas, which could be related to germline BAP1 mutations (2). presenting a baseline mean base of 11.3 mm and a mean Sunlight ultraviolet exposure has been clearly identified as thickness of 5.5 mm (42). The average choroidal melanoma a risk factor for skin melanomas (33), although there is still thickness at diagnosis has exhibited a decreasing trend from debate as to whether this could represent a risk factor for UM: 5.5 mm in the 1970s, to 4.5 mm in the 1990s, to 4 mm in more Some authors support this hypothesis (34), while others refuse recent times (43) This demonstrates the efforts in improving this (35). A previous meta‑analysis revealed that chronic occu‑ the early diagnosis of the tumor. Choroidal melanoma appears pational natural ultraviolet light exposure was a borderline as a pigmented lesion in 55% of cases; in 15% of cases is non‑significant risk factor for UM, whereas geographic latitude non‑pigmented and in 30% of cases has mixed pigmented and and outdoor leisure UV exposure were not significant (35). non‑pigmented features (43). The most common configura‑ Conversely, welding was found to be a significant variable tion of choroidal melanoma is dome‑shaped, in 75% of cases. associated with the development of UM (35). As regards other When the tumor grows, breaking through Bruch's membrane, artificial lights, blue light exposure has been hypothesized it acquires a typical mushroom‑shaped configuration (20% of to play a role in the oncogenetic process and progression of cases) (43). Less commonly, in approximately 5% of cases, UM (36). In addition, occupational cooking seems to be choroidal melanoma presents a diffuse configuration, which associated with a higher risk of developing UM (37). can make diagnosis more challenging (43). Orange pigment and subretinal fluid are typically associated with choroidal 4. Clinical characteristics melanomas. The tumor can cause bleeding with subsequent vitreous hemorrhage, which can obscure the view of the fundus. Patients affected by UM can be asymptomatic (up to 30% of Neovascular glaucoma can develop in advanced cases (43). cases), with this malignancy being an incidental finding at the time of diagnosis (38). Symptoms, when preset, are related 5. Diagnosis to the location of the tumor. Iris melanomas are relatively uncommon (3‑5% of UMs) and diagnosis is mostly secondary The diagnosis of UM is based on a clinical examination and to heterochromia, i.e., changes in iris color, and corectopia, ancillary tests (Table I). The diagnosis of iris melanoma i.e., abnormality in pupil shape, which is present in approxi‑ relies on slit lamp biomicroscopy, anterior segment‑optical mately 45% of cases (10,39). Usually, the tumor is located in coherence tomography (as‑OCT) and ultrasound biomicros‑ the inferior quadrant of the iris (45% of cases) and can cause copy (UBM). In particular, UBM and as‑OCT are helpful secondary glaucoma (direct or indirect obstruction to trabec‑ tools for the assessment of the posterior extension of the ular outflow), ectropion uveae, angle seeding and bleeding tumor (10). Gonioscopy is important to evaluate possible angle with hyphema (10). In some cases, it can be complicated involvement (10). Transillumination may help to evaluate by extraocular extension (3%) (39). Extraocular extension, involvement (10). A thorough fundus examination as well as high intraocular pressure have been shown to be is also required to assess retinal and choroidal condition. The variables associated with metastatic disease (39). Clinically, diagnosis of iris melanoma can be challenging, in particular in an iris melanoma can present several types of configuration cases of small/circumscribed lesions and diffuse melanomas. and levels of pigmentation (from amelanotic to pigmented). In Differential diagnosis includes most commonly, iris nevus, the majority of cases, iris melanoma is circumscribed, while as well as less common lesions, such as cysts, , in a few cases, approximately 10%, it can be diffuse (39,40). leiomyoma, melanocytosis and inflammatory conditions Diffuse iris melanoma is an infiltrative form, undefined and (granulomas) (44,45). The differential diagnosis of diffuse flat, which can prove difficult to diagnose: The presence of melanomas includes diffuse iris nevus, congenital hetero‑ ipsilateral ocular hypertension and acquired heterochromia chromia, congenital, ectropion iridis, melanocytomalytic of the iris are typically associated with this condition (40). A glaucoma, pigmentary glaucoma, siderosis and iridocorneal rare variant of diffuse iris melanoma is the ring melanoma endothelial syndrome (40). Photographic documentation plays of the anterior chamber, a tumor that arises from the angle a relevant role in case of small lesion with a basal diameter and tends to infiltrate the angle structures >360 degrees, with <3 mm, providing information on tumor growth during the a ring pattern of growth (minimal extension towards iris or follow‑up. As reported above, the ABCDEF rule is useful in ciliary body) (41). The main symptom is unilateral glaucoma differentiating an iris nevus from a melanoma. In doubtful and diagnosis is very difficult (depends on careful gonioscopy cases, such as small lesions, fine‑needle biopsy could be and ultrasound biomicroscopy) (41). very helpful in the diagnostic process, with a low risk of When it comes to choroidal and ciliary body melanomas, complications and a good rate of adequate sampling (46). also known as posterior UMs, the main symptoms are blurri‑ The diagnosis of ciliary body melanomas can prove diffi‑ ness (38% of cases), and floaters and flashing lights (7 and 9%, cult when the lesion is small, as the location does not allow for respectively). Less commonly, visual field loss, metamor‑ a good visualization (10). A good scleral indentation during phopsia and pain have been reported (38). The diagnosis of fundus examination could be useful to bring into the view ciliary body melanomas is usually delayed due to their loca‑ these tumors. However, in the majority of cases, tumors are tion and as symptoms tend to appear only when the tumors diagnosed when are large with choroidal or iris invasion (10). are large (10). A study including 492 ciliary body melanomas Transillumination may help to visualize large lesions. A valu‑ found at baseline, a mean tumor base of 11.7 mm and a mean able examination for detecting small ciliary body melanomas thickness of 6.6 mm, proving that the size of these tumors (<4 mm) is UBM, which is useful in follow‑up as well (47). 4 FALLICO et al: INSIGHTS INTO UVEAL MELANOMA , 2013 (104): et al Relevant studies (Refs.) Shields 144 iris melanomas treated with iodine‑125 plaque radiotherapy: 15% years, 7 at recurrence local 1% metastasis rate at 7 years. Posterior UM (including both and choroidal melanoma): ‑COMS trial: Medium choroidal melanoma (2.5‑10 mm apical height tumor basal maximum and diameter ≤16 mm) randomized to iodine‑125 (85 Gy apex dose) or enucleation. No survival differences: 5‑, 10‑ and 12‑year all‑ cause mortality rate was 19, 35 and 43% in the Treatment Type (Refs.) Type ‑Resection (small melanoma) iridotrabeculectomy, iridectomy, iridocyclectomy; ‑Radiotherapy (non‑resectable lesion; seeding): Proton beam good and plaque radiotherapy, local tumor control (102,103); tumors, ‑Enucleation (large poor visual function, recurrent tumors, multifocal melanoma melanoma). and diffuse Posterior UM (including both ciliary body melanoma and choroidal melanoma): Tumors ‑Brachytherapy: <10 mm thickness and <18 mm maximum basal diameter (62); apex dose 70‑100 Gy (105); Ruthenium‑106 lower penetration depth (tumors <6 mm thickness) (106); local recurrence rate: 3% for palladium‑103, 7‑10% for iodine‑125, 14.7% ruthenium‑106 (2); Relevant studies (Refs.) ABCDEF acronym (26) (predictive factors for an iris differentiating melanoma from a nevus): age; B: Blood; Young A: C: Clock‑hour (inferior flat location); D: Diffuse shape; E: Ectropion uveae; F: Feathery margins. ‑In most cases tumors are diagnosed when are large: Baseline mean tumor base mm and mean of 11.7 thickness of 6.6 mm (47).

Tests and imaging (Refs.) Tests ‑Ultrasound biomicroscopy; Anterior (size and posterior extension) (10); ‑Photographic documentation (tumor growth) (10); ‑fine‑needle biopsy (doubtful cases, genetic profile) (58). ‑Ultrasound biomicroscopy: Useful for small melanomas (<4 mm) (47). ‑Ocular ultrasonography: A‑scan) (B‑scan, low‑medium reflectivity/ ultrasonographic hollowness (10,49); ‑Fluorescein and indocyanine green angiography: Progressive hyperfluorescence, ‘double circulation’ pattern (51); Clinical examination (Refs.) ‑Type: Circumscribed tumor (most cases); ‑Type: iris melanoma (10% of cases, diffuse undefined, flat, infiltrative form) (39,40); ring melanoma (rare, angle location with a ring pattern of growth, glaucoma) (41); levels of pigmentation color: Different (from amelanotic to pigmented); diameters; location (inferior quadrant in 45% of cases) (10); associated findings: Heterochromia, corectopia, bleeding, ectropion uveae, extraocular extension, glaucoma (10); Angle involvement (10); ‑Gonioscopy: Ciliary body ‑Transillumination: involvement (10). ‑Anterior segment‑optical coherence tomography (size and posterior posterior extension) (10); ‑Slit lamp examination and dilated fundoscopy with scleral indentation; (10). ‑Transillumination ‑Dilated fundus examination: Configuration dome‑shaped (75% of cases), mushroom‑shaped (5% of (20% of cases), diffuse cases) (43); color pigmented (55% of cases), mixed (30% non‑pigmented (15% of cases) (43); associated features subretinal fluid; orange pigment; bleeding. I. Key points of primary tumor diagnosis and treatment. Table Diagnosis ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ location Tumor Iris melanoma Ciliary body melanoma Choroidal melanoma INTERNATIONAL JOURNAL OF ONCOLOGY 58: 10, 2021 5 , 2017 (116): , 2017 (116): et al Relevant studies (Refs.) and 41% in the enucleation arm, respectively; 5‑, 10‑ and 12‑year metastasis‑related mortality rate (histopathologically confirmed) was 10, 18 and 21% in the brachytherapy arm, 17 and 17% in the and 11, enucleation arm, respectively (72). Brachytherapy ‘as safe as (108); enucleation’ ‑Papakostas by More than 300 patients affected mm (>10 melanoma choroidal large thickness or >16 mm largest basal diameter) treated with proton beam radiotherapy: 70% Eye retention at 10 years; 60% 10‑year mortality (comparable with enucleation); 87% 10‑year local outcome: visual poor control; tumor in 8.7% at 10 years; 20/200 or better 25% neovascular glaucoma. brachytherapy arm, and 19, 35

Treatment Type (Refs.) Type Tumor control and prognosis Tumor comparable to brachytherapy (113); tumor control over 90%, and 5‑year overall survival of preferred to 70‑85% (112); brachytherapy for posterior pole melanomas in large location (112); (>10 mm thickness or >16 diameter) good tumor largest control but risk of ischemic and inflammatory complications (115); ‑Stereotactic radiotherapy (comparable to proton beam) (117); Thickness >10 or ‑Enucleation: 12 mm and/or a basal diameter >18 mm (62,119); ‑Orbital exenteration: Extensive extraocular growth or orbital invasion (10,106); ‑Local resection (exoresection, endoresection): Selected cases. ‑Proton beam radiotherapy: Relevant studies (Refs.) acronym (30) (predictive factors for differentiating a small melanoma from Thickness (>2 mm); a nevus): fluid (subretinal fluid); symptoms; orange pigment (≤3 mm (lipofuscin); margin from optic disc); tumor ultrasonographic hollowness; halo (absent); drusen Thickness ‑TFSOM DIM (54): (>2 mm); fluid (subretinal fluid on optical coherence tomography); symptoms; orange pigment (on autofluorescence), ultrasonographic hollowness, diameter >5 mm (photography). ‑TFSOM UHHD

Tests and imaging (Refs.) Tests ‑Optical coherence tomography: Posterior location; accurate for detecting subretinal fluid; useful for small lesion (50); ‑Photographic documentation: growth, follow‑up; Tumor ‑Fine‑needle biopsy (mainly for genetic profile) (58).

Clinical examination (Refs.) Table I. Continued. Table Diagnosis ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑‑‑‑‑‑‑‑‑‑ location Tumor gamma ray; UM, uveal melanoma. COMS, Collaborative Ocular Melanoma Study; Gy, 6 FALLICO et al: INSIGHTS INTO UVEAL MELANOMA

The diagnosis of choroidal melanoma depends mostly on <2 mm and no other TFSOM UHHD factor, can be consid‑ fundus examination with indirect ophthalmoscopy. The most ered ‘low‑risk’. The presence of one or more TFSOM UHHD important test in the diagnostic process is represented by factors indicates a ‘high‑risk’ nevus (10). Lesions with ≥2 ocular ultrasonography (10,48). In particular, B‑scan ultraso‑ TFSOM UHHD factors are likely to represent small choroidal nography provides information on tumor size and extension. melanomas and treatment should be indicated (29,30). A‑scan ultrasonography provides valuable information on the Recently, TFSOM UHHD has been updated, introducing the reflectivity of the lesion. The presence of acoustic hollowing use of multimodal imaging. The new acronym ‘To Find Small is a typical characteristic of UM (10). Ossoinig considered the Ocular Melanoma Doing Imaging (TFSOM DIM) stands presence of low‑medium reflectivity as one of the cardinal hall‑ for thickness (>2 mm on ultrasonography), fluid (subretinal mark of a melanoma lesion (49). Other A‑scan characteristics fluid on spectral domain‑OCT), symptoms, orange pigment may be a quite regular internal structure with spikes showing (on autofluorescence imaging), melanoma ultrasonographic a similar height or a decreasing height, solid consistency and hollowness, diameter >5 mm (photography). The 5‑year risk sign of vascularization such as a spike showing fast, vertical for transformation of a nevus into melanoma was 22% with two motion with flickering (49). Of note, A‑scan ultrasonography factors, 34% with three factors and 51% with four factors (54). has a limited use in the case of very shallow lesions, with a Therefore, documenting with fundus photograph a choroidal thickness <1.5/2 mm. Clinical examination and information nevus/indeterminate lesion, which appears suspicious, plays a provided by ultrasonography, when carried out by an ocular relevant role for detecting lesion growth during the follow‑up. oncology expert, ensure a high level of accuracy, minimizing Data from the Collaborative Ocular Melanoma Study (COMS) the necessity for biopsy (48). Other useful tests are OCT demonstrated a misdiagnosis rate of approximately 0.5% (55). imaging, fluorescein angiography and indocyanine green angi‑ This finding suggests that the diagnosis of UM can be based ography. Enhanced deep imaging OCT is helpful for studying on clinical examination and tests. However, the COMS applied small lesion (<3 mm in diameter) which are difficult to study strict eligibility criteria, which could have had an influence on with other methods (50). Furthermore, it has a high accuracy the rate of misdiagnosis. Indeed, other studies have found diag‑ in detecting subretinal fluid and may help to differentiate small nostic fine‑needle biopsy necessary in 1‑9% of cases (56,57). choroidal malanomas from other lesion, including nevi (10). The biopsy of intraocular tumors is debated due to the risk However, its use may be limited when it comes to lesions of tumor dissemination, as well as the risk of ocular compli‑ with a thickness >3 mm (10). Fluorescein angiography may cations and inadequate sampling. However, currently, tumor be characterized by a progressive hyperfluorescence which sampling has become more diffuse, usually not for confirming may last for >30‑40 min. In the case of Bruch's membrane the diagnosis, but with the purpose of analyzing the genetic break, the examination can reveal a typical ‘double circula‑ profile for assessing metastatic risk and prognosis (58). For tion’ pattern, due to the presence of tumor vessels underneath choroidal melanomas, fine needle aspiration biopsy (FNAB) the retinal vasculature (51). Indocyanine green angiography is performed using particular precautions to prevent tumor is more useful in showing intra‑lesion vasculature, with the seeding as well as subsequent application of radiotherapy, average peak of hyperfluorescence at 18 min (52). The use of which can help to sterilize seeded cells (58). computed tomography and magnetic resonance imaging with the purpose of studying choridal melanomas has been investi‑ 6. Staging gated (10); however, their application in clinical practice is very limited. The most common differential diagnosis of choroidal The 8th edition of the American Joint Committee on Cancer melanomas is the choroidal nevus. Others can be congenital (AJCC) classification was published in 2016 and provides the hypertrophy of the retinal pigment epithelium (CHRPE), classification of UM as well (59). This is an updated version peripheral eccentric choroidal neovascular membrane, of the 7th edition (60). However, the differences between the choroidal hemangioma, hemorrhagic detachment of pigment two editions are minimal. The widespread T (tumor), N (node), epithelium/ (53). Differentiating a small choroidal mela‑ M (metastasis) staging has been used also for UM. Two clas‑ noma from a choroidal nevus may prove very difficult in some sifications have been developed, one for iris melanoma, and cases. Shields et al provided a mnemonic acronym which can one for choroidal and ciliary body melanoma due to different be useful in daily practice: ‘To Find Small Ocular Melanoma primary tumor staging (T). In either case, T0 refers to cases Using Helpful Hints Daily (TFSOM UHHD), which stands with no evidence of primary tumors and Tx to cases where also for thickness (>2 mm), fluid (subretinal fluid), symptoms, primary tumor cannot be evaluated. Iris melanoma primary orange pigment (lipofuscin), margin (≤3 mm from optic tumor (T) classification is presented in Table II. disc), ultrasonographic hollowness, halo (absent), drusen Primary tumor (T) classification for choroidal and ciliary (absent) (30). Additionally, it has to be taken into account that body melanomas depends on tumor size (thickness and largest a few number of choroidal nevi may transform into choroidal basal diameter), as well as ciliary body involvement and extra‑ melanomas (<1:8,000) (28). When one of the TFSOM UHHD ocular extension (59). Primary tumor classification according factor is present, there is a 38% risk for the lesion to transform to tumor size is displayed in Table III. into melanoma at 5 years, increasing to 50% when at least All T values can be featured by a letter from ‘a’ to ‘d’, two factors are present (29,30). If the lesion has the following where ‘a’ indicates nor ciliary body involvement neither extra‑ 3 TFSOM UHHD factors, such as a thickness of >2 mm, a ocular extension, and ‘b’ indicates ciliary body involvement location close to the disc and symptoms, the risk for transfor‑ without extraocular extension; ‘c’ indicates no ciliary body mation into melanoma at 5 years increases to 69% (29,30). A involvement, but a ≤5 mm extraocular extension; and ‘d’ indi‑ choroidal nevus with drusen (signs of chronicity), a thickness cates both ciliary body involvement and a ≤5 mm extraocular INTERNATIONAL JOURNAL OF ONCOLOGY 58: 10, 2021 7

Table II. Iris melanoma primary tumor (T) classification according to the American Joint Cancer Committee (AJCC 8th edition) (59).

Primary tumor (T) classification Explanation Sub‑stages

T1 Tumor limited to the iris T1a: not >3 clock hours in size T1b: >3 clock hours in size T1c: T1 with secondary glaucoma T2 Tumor confluent with or extending T2a: Confluent with or extending into the ciliary into the ciliary body, choroid, or both body, without secondary glaucoma T2b: Confluent with or extending into the ciliary body and choroid without secondary glaucoma T2c: Confluent with or extending into the ciliary body, choroid, or both, with secondary glaucoma T3 Tumor confluent with or extending into the ciliary body, choroid, or both, with scleral extension T4 Tumor with extrascleral extension T4a: Extrascleral extension ≤5 mm in diameter T4b: Extrascleral extension >5 mm in diameter

The information presented in the table is derived from a previous study (59).

Table III. Primary tumor (T) classification for choroidal and ciliary body melanoma based on thickness and largest diameter (59).

Largest basal diameter, mm ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ Thickness ≤3 3.1‑6 6.1‑9 9.1‑12 12.1‑15 15.1‑18 >18

≤3 mm 1 1 1 1 2 2 4 3.1‑6 mm 1 1 1 2 2 3 4 6.1‑9 mm 2 2 2 2 3 3 4 9.1‑12 mm 3 3 3 3 3 3 4 12.1‑15 mm 3 3 3 3 3 4 4 >15 mm 4 4 4 4 4 4 4

The information presented in the table is derived from a previous study (59).

extension (59). Additionally, the T4e category includes any Spindle cell UM (>90% spindle cells); epithelioid cell UM tumor size with an extraocular extension of >5 mm (59). (>90% epithelioid cells); mixed UM, consisting of <90% Regional lymph nodes (N) include preauricular, subman‑ spindle cells and >10% epithelioid cells (59). Spindle cells dibular and cervical sites. Node assessment applies to tumors feature ovoid nuclei and their growth exhibits a compact with extrascleral growth and conjunctival involvement. Nx and cohesive pattern. Epithelioid cells are pleomorphic, includes cases where nodes cannot be evaluated; N0 indicates with larger and irregular shape compared to spindle ones. absence of node metastasis; and N1 indicates the presence of The growth pattern of their nuclei and nucleoli is less node metastasis or discrete tumor deposits in the orbit; the N1 cohesive compared to spindle type; their cytoplasm is acido‑ stage is classified into N1a (metastasis in one or more regional philic (59). Gx indicates cases where the grade cannot be lymph nodes) and N1b (no positive regional lymph nodes, but evaluated. G1 includes spindle cell UM; G2 mixed cell UM; the presence of discrete tumor deposits in the orbit that are G3 epithelioid cell UM (59). not contiguous to the eye). Distant metastases are evaluated At the time of diagnosis, it is mandatory to carry out in the ‘M’ category: M0 indicates no metastasis; M1 indicates imaging tests to identify systemic metastases, as the presence the presence of distant metastasis (‘a’ ≤3 cm metastasis; ‘b’ of metastases has a relevant effect on the management plan. 3.1‑8 cm metastasis; ‘c’ >8.1 cm metastasis) (59). The AJCC In the past, baseline imaging consisted of abdominal ultraso‑ anatomic stage is presented in Table IV. nography and chest radiography. Given the low sensitivity of A further classification depends on the histological grade those tests (61), baseline modern imaging work‑up for ruling (G) of the tumor. Basically, there are three histopathologic out metastases includes usually one of the following protocols: types of UMs, according to their cytological composition: Computed tomography (CT) of chest and abdomen; chest CT 8 FALLICO et al: INSIGHTS INTO UVEAL MELANOMA

Table IV. Anatomic stage according to AJCC cancer staging Several factors have been investigated with the purpose manual, 8th edition (59). of assessing their possible influence on the prognosis of UMs, and the role of some is still debated (10). For instance, Stage T N M whether age and sex may have an influence on the prognosis is not yet completely clear. It seems that a younger age may I T1a N0 M0 exert a protective effect against metastatic disease as the IIA T1b‑d N0 M0 immune response is more robust, lesions tend to be smaller T2a N0 M0 and genetic mutations are less common compared to older IIB T2b N0 M0 aged patients (17,66). In a study including >8,000 patients with T3a N0 M0 melanoma, Shields et al (69) reported a cumulative rate of metastasis at 10 year of 9% in patients <20 years of age, 23% IIIA T2c‑d N0 M0 in patients 21‑60 years of age and 28% in patients >60 years of T3b‑c N0 M0 age; the cumulative 10‑year mortality rate was 5% in patients T4a N0 M0 <20 years of age, 11% in patients 21‑60 years of age and 16% in IIIB T3d N0 M0 patients >60 years of age; at 10 years, metastases were found in T4b‑c N0 M0 0% of patients 0‑10 years of age, 10% of patients 11‑20 years of IIIC T4d‑e N0 M0 age, 21% of patients 41‑50 years of age, and in 30% of patients 71‑80 years of age. The authors concluded that young patients IV Any N1 M0 had a lower rate of metastatic disease. However, the proportion Any N1 M1a‑c of iris melanoma was 21% in young (≤20 years old), 4% in mid adult (21‑60 years old) and 2% in older adult (>60 years The information presented in the table is derived from a previous study (59). old) patients (69). Whether the female sex could have a better prognosis compared to the male sex remains controversial: In one study, the mortality rate at 10 years was found to be 2‑fold greater in males compared to females, and time to and magnetic resonance imaging (MRI); whole body develop metastatic disease was shorter in males compared to positron emission tomography‑CT (62). females (metastatic disease at 5 years from diagnosis of UM in 84% of males compared to 50% of females) (70); however, no 7. Prognosis sex‑related differences in survival analysis were found in the COMS study (71). UM presents a high mortality rate and up to 50% of cases Tumor size has been demonstrated to have a significant metastasize (2,63). A large study including both iris and poste‑ effect on the development of metastases. In a large study rior melanomas revealed a metastatic rate of 15% at 5 years and including both iris and posterior UM, the 10‑year metastasis 25% at 10 years (42). The most common metastasis site is the rate was 6% for a thickness of 0‑1 mm, 12% for a thickness of liver (60‑89%), followed by the lungs (24‑29%), skin and soft 2.1‑3 mm, 16% for a thickness of 3.1‑4 mm, 27% for a thick‑ tissue (11‑12%), bone (8‑17%) and lymph nodes (11%) (63,64). ness of 4.1‑5 mm, 41% for a thickness of 7.1‑8 mm, and 51% The prognosis of UM has been related to several variables. for a thickness of >10 mm (42). A hazard ratio of 1.06 was First, location has been shown to have an influence on prog‑ found for a 1 mm increase in thickness (42). The COMS report nosis. Iris melanoma has a mortality rate 5‑10‑fold lower than on the mortality outcome of medium choroidal melanomas posterior UM. In a review article of >8,000 cases of UM, the (2.5‑10 mm thickness and ≤16 mm largest basal diameter) 10‑year metastasis disease was shown to be 33.4% for ciliary revealed a similar rate of melanoma metastasis‑related body melanomas, 25% for choroidal melanomas and 6.9% for mortality at 10 years in both brachytherapy and enucle‑ iris melanomas (42). The better prognosis of iris melanoma ation arms (18 and 17%, respectively), and depicted a larger can be explained by factors including a lower biologic activity, maximum basal tumor diameter as a primary predictor of younger age and smaller size (65,66). The cumulative propor‑ melanoma metastasis‑related death (72). Likewise, the COMS tion of metastatic disease and mortality at 5 years has been report on large choroidal melanomas (>10 mm thickness and found to be 5.2 and 2.2%, respectively, increasing to 8.8 and >16 mm diameter) revealed a melanoma metastasis‑related 3.3% at 10 years, respectively (39). In the case of iris mela‑ mortality of 40% in the enucleation arm at 10 years, and noma, factors predicting melanoma‑related metastasis are an depicted a larger maximum basal tumor diameter as a primary older age, increased thickness, secondary glaucoma, angle predictor of melanoma metastasis‑related mortality (71). A involvement and extraocular extension (39,42,67). A study previous meta‑analysis on choroidal melanomas treated with investigating the very long‑term prognosis of patients with enucleation reported a 5‑year mortality rate of 16% in the case posterior UM reported a melanoma‑related mortality rate of tumors with a thickness of <2 or 3 mm and a basal diam‑ of 31% at 5 years, 45% at 15 years and 49% at 25 years (68). eter <10 or 11 mm, 32% in case of tumors with a thickness of The COMS study, which included choroidal melanomas and 3‑8 mm and a basal diameter <15 or 16 mm, and 53% in case featured strict eligibility criteria excluding peripapillary tumors of tumors with a thickness of >8 mm and a >16 mm basal and predominately ciliary body tumors, reported a cumulative diameter (73). metastasis rate of 25% at 5 years and 34% at 10 years (64). Not surprisingly, the AJCC staging has been demonstrated Following the development of metastasis, the mortality rate to play a prognostic role. At the 10‑year follow‑up, tumors with was 80% within one year and 92% within two years (64). a T1 stage had a 15% metastatic rate, increasing to 25% for INTERNATIONAL JOURNAL OF ONCOLOGY 58: 10, 2021 9

T2 tumors and to 49% for T3 tumors. Melanomas with a T4 and BAP1 mutation has been found to be a prognostic factor stage presented a 63% metastatic rate (74). As reported above, for metastatic disease (32). The partial or complete loss of the location of the melanoma has a noticeable relevance in chromosome 1p predicts a negative prognosis. It is usually terms of metastatic risk. Overall, ciliary body melanoma could associated with 3, but can also occur alone (76). be considered the most aggressive. Ciliary body melanomas Concomitant monosomy 3 and chromosome 1p loss is by far a present a 2 to 4‑fold higher risk of metastasis than choroidal stronger predicting factor for metastatic disease compared to ones (75). The possible reasons for this higher tendency to the loss of either chromosome 3 or chromosome 1p (81). The metastasize may be related to the delay in diagnosis (ciliary most common alteration affecting is a gain in body tumors are less symptomatic and, usually, are large at chromosome number. Indeed, chromosome 8q gain has been time of diagnosis), the relevant vascularization of ciliary body, found in 41‑53% of UM cases, whereas a loss of chromosome and the higher incidence of chromosomal predisposing altera‑ 8q is rare (76). Similar to chromosome 1p loss, chromosome tions (10,76). The presence of oculodermal melanocytosis 8q gain can occur alone or in combination with monosomy 3. represents a risk factor for the development of UM, as well as Chromosome 8q gain in combination with monosomy 3 has for metastasis development in patients affected by UM (76). been associated with a poorer prognosis compared to each Other tumor‑related features that may affect prognosis are alteration alone: The 5‑year mortality rate has been reported ‘diffuse’ configuration and extraocular extension. The risk to be 31% in cases of 8q gain, 40% in cases with monosomy of melanoma‑related metastasis has been demonstrated to be 3, 66% in cases of concomitant 8q gain and monosomy 3 (82). higher in diffuse iris melanoma and diffuse choroidal mela‑ Conversely to chromosome 8 gain, gain is noma compared to non‑diffuse iris and choroidal melanomas, a predictor of a good prognosis and tends to be mutually respectively (76). Extraocular extension has a significant exclusive with monosomy 3 (76). The occurrence of both negative effect on prognosis when >5 mm: The mortality rate monosomy 3 and chromosome 6 gain has been reported only at 5 years has been shown to be 37, 24 and 78% for patients in 4% of UM (83). On the contrary, chromosome 6 loss is a with microscopic, small (1‑4 mm) and large extrascleral exten‑ predictor of an unfavorable prognosis: The loss of 6q has been sion, respectively (77). Histopathology has been shown to found in 40% of tumors with metastatic disease vs. 7% of play a relevant role in prognostication. As regards cell type, metastasis‑free melanomas (84). the optimal prognosis has been associated with spindle cell Over the past years, considerable efforts have been made melanoma, the worst with epithelioid cell type, while mixed to improve epigenetic and transcriptomic analyses. Gene type has an intermediate prognosis (76). UM cases with a high expression profiling has provided a prognostic classification of mitotic activity have a worse prognosis compared to those with UM. This classification consists of two main classes: Class I a low mitotic activity (78). Nucleoli size is another histopatho‑ melanomas associated with a low risk of metastasis develop‑ logic variable which affects prognosis. The mean diameter of ment and class II melanomas associated with a high risk of the 10 nucleoli with the largest size (MLN) is used for prog‑ metastatic development (85). These results were based on nostication. A large MLN predicts a poor prognosis (76). Of the analysis of the mRNA expression of 15 genes (12 target note, epithelioid cells are characterized by larger MLN, but genes and 3 controls) and have been validated in a clinical MLN has been shown to be an independent factor as well (79). setting (86). A test analyzing these 15 genes is available and Tumor vascularity also has an influence on prognosis. A can be used in clinical practice with ease, on samples obtained high microvascular density, as well as specific microvascular from enucleation, tumor resection and FNAB (87). Class I can patterns, such as the presence of networks or loops, have been be divided into class IA with a 2% metastatic risk at 5 years identified as predictors of a worse prognosis (76). An unfavor‑ and class IB with a 20% metastatic risk at 5 years. Class II able prognosis has also been associated with the presence of presents a 72% metastatic risk at 5 years (88). These data allow numerous tumor‑infiltrating macrophages, high insulin‑like patients to be offered a personalized management based on risk growth factor‑1 receptor expression, and a high expression of stratification (88). A further point that needs to be mentioned human leukocyte antigen (HLA) class I and II (76). with regards to prognostication is the concept of micrometas‑ Recently, when it comes to prognostication, further atten‑ tasis. Eskelin et al investigated the metastasis doubling time tion has been paid to cytogenetic characteristics. Usually, and postulated that micrometastases could begin up to 5 years genetic tests are carried out on samples obtained from FNAB prior to primary tumor treatment (89). Taking into account all or enucleation specimens. Most relevant cytogenetic altera‑ these considerations, the early diagnosis and treatment of UM, tions include chromosome 3, 1, 8 and 6 (76). Chromosome 3 including small melanomas, may represent a key strategy for a loss represents a predictor of a poor prognosis. In particular, positive long‑term prognosis (10). the complete loss of one chromosome 3, known as mono‑ somy 3, has been identified as the most relevant prognostic 8. Primary tumor treatment factor, being associated with an increased risk of metastatic disease (76). In a series of 54 UMs, monosomy 3 was found The management of UM represents a multi‑disciplinary chal‑ in 56% of cases. Those with monosomy 3 presented a 3‑year lenge, involving a variety of physicians specialized in ocular mortality rate of 50 vs. 0% of those without monosomy 3 (80). oncology, such as ophthalmologists, radiologists, medical and Monosomy 3 was found in association with other unfavorable radiation oncologists (90). It is important to note that despite prognostic factors, such as epithelioid type, vascular loops, improvements being made in primary tumor treatment, the high mitotic activity, extrascleral extension, ciliary body metastasis rate and overall survival has remained unaltered location and a large diameter (76). Furthermore, BAP1 has over the past decades (4,91). Once metastatic disease has been been located on the short arm of this chromosome (3p21.1) diagnosed, the overall survival is as low as roughly one year, 10 FALLICO et al: INSIGHTS INTO UVEAL MELANOMA as shown by a recent meta‑analysis (91). In fact, patients who radiotherapy, including plaque brachytherapy or external beam present with metastasis at the diagnosis of the primary tumor radiation therapy, mostly used for small/medium melanomas, often do not undergo the aggressive treatment of the primary and enucleation surgery, mostly used for large melanomas and tumor (62). Primary UM treatment can be divided into two poor visual function (10). Other possible treatment options types: Globe‑preserving treatment and enucleation (Table I). include surgical resection and laser treatments, such as trans‑ The former one includes radiation therapy, laser and surgical pupillary thermotherapy and photodynamic therapy (PDT) (2). therapy. For many years the only available treatment for UM Importantly, in the case of indeterminate lesions, which can was enucleation. In the 1970s, efforts were made to develop be either a choroidal nevus or a small melanoma, an observa‑ globe‑preserving alternatives (92). Subsequently, with the tion can represent the first approach: The patient is monitored introduction of radiation therapy, there has been a shift towards for documented growth or TFSOM UHHD (30) risk factors a globe‑sparing approach rather than enucleation surgery, in (as reported above). If there is evidence of documented growth particular since the COMS study revealed comparable survival or the presence of TFSOM UHHD (30) factors, treatment rate between enucleation and plaque radiotherapy in patients should be considered (62). In some selected patients affected with medium choroidal melanomas (72). Radiotherapy tends by small choroidal melanomas (thickness of <3 mm and largest to be the preferred treatment for small and medium UMs, basal diameter <10 mm), usually presenting with low‑grade whilst enucleation is usually performed for larger and more tumor (stable or growing slowly), an advanced age, multiple advanced melanomas (10). Tumor characteristics, as well comorbidities and limited life expectancy, observation can as patient characteristics must be taken into account when represent an alternative to the treatment. However, patients selecting the appropriate treatment. must be informed about both the risks of treatment (visual Iris melanoma treatment depends on the size of the lesion, loss) and the risk of metastasis (unquantified albeit small) for as well as on its characteristics. A small lesion with a basal observation (62,105). diameter <3 mm, with no other sign and no symptoms, that Radiotherapy for UM includes plaque brachytherapy, may be a nevus or a small melanoma (indeterminate lesions), proton beam radiotherapy and photo beam radiation can be monitored periodically with photographic documenta‑ therapy (stereotactic radiotherapy). Radiotherapy has gained tion for evaluating possible growth (10). Small circumscribed increasing popularity for the treatment of UM and has lesions with documented growth can be treated with sector replaced enucleation surgery for melanoma of suitable size and iridectomy (93). If there is an involvement of the anterior location (62). It is a globe‑preserving treatment, which ensures chamber, a portion of the trabecular meshwork needs to be excellent local tumor control (2). Following the introduction removed as well; this type of resection is termed iridotrab‑ of radiotherapy for the treatment of UM, the main concern eculectomy. If there is an involvement of the ciliary body, of physicians was whether there was a difference in survival an iridocyclectomy can be performed, resecting a portion between radiotherapy and enucleation (106). Therefore, from of the iris and ciliary body (10). Intraocular surgery can be 1986 to 2003, the COMS group conducted two large multi‑ associated with complications, such as hypotony, retinal center clinical trials comparing survival between radiotherapy detachment, lens subluxation, phthisis, endophthalmitis and and enucleation in patients affected by medium and large sympathetic ophthalmia (93‑95). Larger melanomas are choroidal melanoma (72). Patients affected by large choroidal usually non‑resectable and treatment is based on radiotherapy melanoma (apical height >10 mm and maximum basal tumor or enucleation (10,96‑98). In 1955, Lloyd and Ellis described diameter >16 mm) were randomized to enucleation alone the use of radioactive wires (tantalum), inserted into the eye, or external beam irradiation (20 G) preceding enucleation for the treatment of small iris melanoma (99). Subsequently, surgery; patients affected by medium choroidal melanoma external beam and plaque radiotherapy became available (2.5‑10 mm apical height and maximum basal tumor diam‑ for the treatment of iris melanomas. Anterior segment irra‑ eter ≤16 mm) were randomized to iodine‑125 brachytherapy diation can be beneficial for non‑resectable lesions and for or enucleation (107). The COMS was the largest randomized extensive seeding, as treatment margins are larger compared controlled trial (RCT) performed in ocular oncology, with to simple resection (100,101). In a small series of patients >2,000 patients enrolled (107). The results revealed no survival with iris melanoma, it has been shown that proton beam and differences at 5, 10 and 12 years between plaque brachytherapy plaque radiotherapy can achieve local tumor control in up to and enucleation in patients with medium choroidal melanoma: 93% (102) and 97% (103) of cases, respectively. A larger study The 5‑, 10‑ and 12‑year all‑cause mortality rate was 19, 35 on 144 patients reported local recurrence in approximately and 43% in the brachytherapy arm, and 19, 35 and 41% in the 15% of cases at 7 years, showing an adequate local tumor enucleation arm, respectively; the 5‑, 10‑ and 12‑year metas‑ control; metastasis rate at 7 years was 1% (104). Even if radia‑ tasis‑related mortality rate (histopathologically confirmed) tion treatment is a globe‑spearing approach, complications was 10, 18 and 21% in the brachytherapy arm and 11, 17 and may be severe and sight‑threatening, including corneal opaci‑ 17% in the enucleation arm, respectively (72). This reassured ties, cataract and iris neovascularization, culminating in vision that brachytherapy is ‘as safe as enucleation’ (108). However, loss (10,101). Enucleation surgery is usually reserved for large in a number of cases, metastases developed in a very short tumors, poor visual function, recurrent tumors, multifocal iris amount of time, suggesting that systemic spread was present melanoma and diffuse iris melanoma (10). However, radiation at the time of primary lesion treatment; this could have led to therapy has recently exhibited good local tumor control also a lack of statistical power (108). Nonetheless, the conclusion for both diffuse and multifocal iris melanomas (100,101). of comparable efficacy in terms of survival between brachy‑ The treatment of posterior UM can be surgery, radiation therapy and enucleation could be considered correct (106). As therapy or laser. In general, the most common treatments are regards the large choroidal melanoma arms, no differences in INTERNATIONAL JOURNAL OF ONCOLOGY 58: 10, 2021 11

5‑ and 10‑year tumor‑related mortality were found between for UM, with a rate of local tumor control >90%, and a 5‑year enucleation alone and enucleation with preoperative irradia‑ overall survival of 70‑85% (112). Charged‑particle radio‑ tion (71,107); this finding confirmed that primary enucleation therapy could be preferred to brachytherapy for tumors with alone does not increase mortality from metastatic disease as a location that may challenge plaque positioning, with also a was hypothesized by Zimmerman et al (109). risk of suboptimal immobilization of the plaque (for instance, Brachytherapy is one of the most largely used conserva‑ posterior pole) (112,114). Proton beam radiotherapy can also tive treatments for UM (92). Following the publication of be used for the treatment of large tumors; however, it could COMS reports, brachytherapy has become the treatment of be challenged by a high rate of local recurrence and a high choice for suitable tumors (62). Brachytherapy is used for risk of radiation‑induced complications that can lead to vision posterior UMs with a thickness of <10 mm and a maximum loss and/or secondary enucleation. Additionally, a ‘toxic tumor basal diameter<18 mm. Selected iris melanomas and ciliary syndrome’ has been described following radiotherapy for large body melanomas (<10 mm thickness and no extensive circum‑ tumors as a result of severe intraocular , which ferential growth) can be considered for brachytherapy as causes exudative and ischemic complications (115). A previous well (62). The radiation dose delivered to the tumor apex is study on >300 patients affected by large choroidal melanoma 80‑100 Gy (10). According to the 2014 consensus guidelines (>10 mm thickness or >16 mm largest basal diameter; >8 mm from the American Brachytherapy Society, the apex dose can thickness in case of optic nerve involvement) demonstrated range from 70‑100 Gy (105). The apex dose in the COMS that proton beam radiotherapy allowed patients to retain the trial was 85 Gy (110). The plaque features a saucer shape and eye in 70% of cases at 10 years; the 10‑year mortality (60%) contains the radioisotope. The plaque is sutured to the sclera was comparable with enucleation; and the 10‑year local (positioned corresponding to the tumor) until the dose has tumor control was 87% (116). However, visual outcome was been delivered. Plaque size has to physically exceed tumor poor, with only 8.7% of cases with a visual acuity of 20/200 margin by at least 2 mm (free‑margin) (106). During the or better at 10 years; additionally, 25% of cases developed surgery, the plaque has to be positioned adequately, in rela‑ neovascular glaucoma; this rate increased to roughly 35% by tion to tumor location: Intraoperative US or transillumination 5 years following treatment (116). Stereotactic photon beam are used to ensure a proper positioning; a notched plaque radiotherapy with gamma knife, cyber knife or linear accel‑ is used in case of juxta‑papillary lesions (106). The most erator, delivers high doses from multiple directions, trying largely used radioisotopes are iodine‑125, ruthenium‑106 to spare surrounding tissues (106). Tumor control, survival and palladium‑103 (62). Ruthenium‑106 emits beta radia‑ outcome and visual outcome have been reported comparable tion, while iodine‑125 gamma radiation. Beta radiation has with those of proton beam radiotherapy (117). a lower depth of penetration compared to gamma radiation. Common sight‑threatening complications from radio‑ As a consequence, ruthenium‑106 can be used for tumor with therapy are radiation retinopathy, known as radiation a thickness <6 mm (2,106). The advantage of this limited maculopathy when affecting the macula, and radiation papil‑ depth of penetration should be a reduced damage to eye lopathy, when affecting the optic disc. Radiation maculopathy structures (92) A previous study on 400 eyes treated with and optic nerve atrophy can lead to visual loss (118). These palladium‑103 plaque, revealed a favorable visual outcome complications are related to the radiation‑induced damage to and local tumor control compared with ruthenium‑106 and the retina and optic nerve. Tumor size and location, as well iodine‑125; the mean apex dose was 73.3 Gy (for an equiva‑ as dosimetric parameters have an influence on their develop‑ lent dose, more radiation was delivered in palladium‑treated ment (112,118). For instance, the cumulative incidence rate tissue compared to iodine‑treated) (111). The local recurrence of radiation maculopathy has been reported as high as 64% rate has been reported as 3% for palladium‑103, 7‑10% for at 5 years for tumors located within 4 disc diameters to the iodine‑125 and 14.7% for ruthenium‑106 (2). Local recur‑ macula (118). Other complications of radiotherapy include rence can be either re‑treated with brachytherapy or treated glaucoma, neovascular glaucoma, cataract, vitreous bleeding, with enucleation. A further therapeutic option suitable for ocular surface problems, radiation‑induced dry eye, keratitis, minimal margin recurrence can be transpupillary thermo‑ diplopia/strabismus and scleral necrosis (10,106,112). therapy (TTT) (62). Proton beam radiotherapy delivers high Enucleation represented the mainstay treatment for UM dose radiation by using charged particles and relatively prior to the advent of radiotherapy (92). Thereafter, enucleation sparing superficial tissues (106). Proton beam radiotherapy has been the second most common treatment for UM (119). In can be used for the treatment of both posterior UMs and iris the 1970s, a concern as to whether enucleation could increase melanomas (96,112). Tantalum markers are sutured to the the risk of metastasis was raised due to the diffusion of the sclera and their distance from tumor margins, limbus and from ‘Zimmerman hypothesis’, which based this assumption on the each other, is measured for proper localization and treatment potential dissemination of tumor cells into the blood system planning. Usually, a 2‑mm safety margin is used. Following at the moment of optic nerve cutting (109). As reported above, a simulation phase, treatment is delivered in 4 consecutive this hypothesis was disproved following the publication of the day‑sessions, with a total dose of 56 Gy (106). In the past, the COMS findings. Indications for enucleation include the pres‑ total dose amounted to 60‑70 Gy, whereas more recent studies ence of a large tumor, poor visual potential and extraocular have used a dose ranging between 50 and 60 Gy (112). Proton growth (10,114). Enucleation is the preferred treatment for beam radiotherapy for posterior UM presents comparable tumors with a thickness >10 or 12 mm and/or a basal diameter outcomes in terms of tumor control, systemic prognosis and >18 mm (62,119). For these lesions, charged‑particle radio‑ visual result compared to brachytherapy (113). Proton beam therapy can still be offered, although high‑dose irradiation radiotherapy is considered as an effective and safe treatment carries a high risk of complications that can lead to vision loss 12 FALLICO et al: INSIGHTS INTO UVEAL MELANOMA and, possibly, to eye loss; furthermore, these patients have to Laser treatment for posterior UMs includes TTT and cope with the anxiety related to possible recurrence (62,119). PDT. TTT delivers an infrared laser light (810 nm) to the In a series of 1,632 patients treated for UMs from 1993 to 2002, tumor surface through a dilated pupil. The laser causes an 35% underwent primary enucleation, 31% plaque brachy‑ increase in tumor temperature, heating its cells to 45‑60˚. therapy and 17% proton beam radiotherapy. Factors associated As a result, tumor abnormal vessels are obliterated, leading with primary enucleation were tumor size, proximity to the to a necrotic process (130). This treatment can be used for optic disc, extensive involvement of iris, angle or ciliary small choroidal melanomas (thickness ≤3 mm), due to limited body (120). Orbital exenteration can be required in the case of laser penetration (maximum penetration of 4 mm) (130). extensive extraocular growth or orbital invasion (10,106). Tumor pigmentation may also have an influence on treatment The local resection of the tumor represents a globe‑sparing outcome because amelanotic lesions feature poor heat absorp‑ surgical treatment which may be suitable for selected patients tion (131). Therefore, small pigmented lesions may be suitable affected by posterior UMs. Tumor resection can be based for TTT. However, the use of primary TTT as a sole treatment either on an external approach, known as exoresection, or of small choroidal melanomas has been questioned due to high on an ab‑interno approach, known as endoresection. The rate of local recurrence, reported up to 29% of cases (132). As exoresection of a posterior UM needs to be performed under a consequence, TTT should be preferably used in combina‑ hypotensive general anesthesia due to the high hemorrhagic tion with radiotherapy (106). A study including 143 patients risk (106). A lamellar, partial thickness, scleral flap is created with choroidal melanoma compared brachytherapy alone around the lesion, which is ‘en‑bloc’ excided together with the vs. brachytherapy combined with TTT: Combined treat‑ inner scleral layer. The superficial scleral flap is, then, used to ment provided a lower recurrence rate, while the metastasis close the deep opening (121‑124). This surgery is not commonly rate and overall survival were comparable (133). However, a performed and can be associated with sight‑threating compli‑ larger study including 449 patients with choroidal melanoma cations, such as bleeding, , cataract and revealed no difference in tumor control and vital prognosis tumor recurrence. Shields et al reported outcomes of 95 poste‑ between brachytherapy and brachytherapy combined with rior UMs treated with exoresection: Retinal hemorrhage, TTT; of note, brachytherapy alone provided a better visual retinal detachment and tumor recurrence/residual occurred in outcome (134). The treatment of large UMs with proton beam 35, 28 and 15% of cases, respectively (121). Adjuvant plaque radiotherapy combined with TTT could reduce the number of brachytherapy can be associated with exoresection to reduce secondary enucleations (135). It is worth mentioning a large the risk of recurrence (125). Recently, Caminal et al (126) study on 391 patients with choroidal melanoma treated with described the outcomes of transcleral resection performed primary TTT: Tumor recurrence occurred in 28% of patients without hypotensive anesthesia and combined with vitrec‑ and its predictive factors were the presence of ocular symp‑ tomy with silicone oil and plaque brachytherapy. The authors toms, proximity to the optic disc, subretinal fluid, greater concluded that this demanding procedure, when successful, thickness and elevation of post‑treatment tumor scar (130). could provide a better visual outcome compared to plaque Common complications following TTT for UM were retinal brachytherapy. However, submacular hemorrhage, retinal vein occlusion (26%), macular epiretinal membrane (23%), detachment and ocular hypertension occurred in 16, 21 and macular edema (9%) and vitreous hemorrhage (10%) (130). In 21% of cases, respectively (126). Endoresection involves the general, TTT or PDT are used only if the lesion is very small piecemeal removal of the tumor by using a vitreous cutter and there is a high risk of visual loss from radiotherapy (106). during a pars plana vitrectomy (127,128). Tumor residual Of note, TTT represents a treatment option in case of tumor can be destroyed using endolaser photocoagulation. Silicone recurrence after brachytherapy (136). oil is used as an endotamponade. The timing of silicone oil PDT is a non‑thermal laser treatment which involves removal is variable, ranging from 3 to 8.8 months (127,128). the administration of a photosensitizer activated with laser Due to concerns regarding tumor seeding during surgical light (137). This minimally invasive therapy has also been manipulation, adjuvant radiotherapy has been associated described for the treatment of ocular tumors, including with endoresection surgery (128,129). Endoresection has choroidal melanoma (137). Following the intravenous admin‑ been proposed to treat posterior UMs with a juxta‑papillary istration of the photosensitizer and its accumulation into the location, as radiotherapy is likely to cause radiation‑induced tumor tissue, laser light is delivered to activate the photosen‑ optic neuropathy (127). Konstantinidis et al (127) reported on sitizer. This, once activated, it exerts a direct cytotoxic effect 71 patients with juxta‑papillary UM treated with endoresec‑ on the tumor, causing peritumoral vasculature destruction and tion. Over a median follow‑up of 4 years, local recurrence local inflammation, with subsequent autophagy (137). It is occurred in 3% of cases, and retinal detachment in 22% of important to highlight that the presence of pigmented tumor cases. All‑cause mortality was 9 and 20% at 5 and 10 years, is a contraindication for PDT (137,138). Pigmentation seems respectively. The authors concluded that the procedure could to prevent light penetration into the lesion (137). Thus, PDT be a useful alternative to irradiation for juxta‑papillary mela‑ could be used for the treatment of small amelanotic melanomas nomas (127). Endoresection has been used also in the treatment (<4 mm thickness) (10). Most commonly, PDT is performed of large UMs, in combination with preoperative stereotactic using verteporfin as a photosensitizer. Verteporfin PDT for radiotherapy and adjuvant plaque brachytherapy (128). Over a choroidal melanoma treatment can be with either standard mean follow‑up of 32 months, 15% of cases required a further fluence (50 j/cm2 in 83 sec) or a double fluence (100 j/cm2 in vitrectomy, mainly due to retinal detachment, 5% of cases had 166 sec) (137). A previous review article of 6 reports including a local recurrence, and 15.5% of cases died from metastatic a total of 38 choroidal melanoma cases primarily treated disease (128). with verteporfin PDT showed 80% tumor control over INTERNATIONAL JOURNAL OF ONCOLOGY 58: 10, 2021 13

31 months (137). A more recent study on 12 eyes with choroidal To date, no therapy has been demonstrated to be effective melanoma reported tumor control in 67% of cases, while 33% for the treatment of metastatic disease in patients with UM. failed to regress (138). PDT with indocyanine green has been Metastatic UM is associated with a poor prognosis. Several used for the treatment of choroidal melanoma, exhibiting a chemotherapeutic drugs, including dacarbazine, cisplatin, high rate of tumor control (137). Notably, a previous study on trosulfan, temozolomide and fotemustine, have been inves‑ 25 patients demonstrated good local tumor control combining tigated, exhibiting a low response rate and disappointing both TTT and indocyanine green PDT for the treatment of outcomes (114,147). While immunotherapy has noticeably small and medium choroidal melanomas (139). improved outcomes in metastatic cutaneous melanoma, this has not been the case for metastatic UM (148). A possible 9. Surveillance and metastatic disease treatment reason for such a different response to immunotherapy could be related to the different biological characteristics between Following primary tumor treatment, patients with UM need to cutaneous and UMs, as well as their different immunoge‑ receive periodical ocular and systemic surveillance. The aim nicity (149,150). Ipilimumab has shown a response rate of of ocular follow‑up is the early detection and management of approximately 5‑10%, with an overall survival ranging from 6 possible local tumor recurrence and treatment‑related compli‑ to 9.7 months (147). Nivolumab has shown in a prospective trial cations. In general, close follow‑up visits are scheduled in the a 6% response rate and an overall survival of 11 months (151). A early post‑operative period, which, then, are extended to a previous retrospective study reported outcomes of 89 patients 3‑ to 6‑month interval for a few years; thereafter, if the clinical treated with ipilimumab plus nivolumab, showing a 11.6% condition is stable, follow‑up can be arranged each year (106). response rate and an overall survival of 15 months (152). Local tumor recurrence is usually managed in the same manner The understanding of the molecular mechanisms involved as the primary tumor (106). The treatment of secondary orbital in UM carcinogenesis and progression will contribute to the involvement is challenging and includes radiotherapy and development of targeted therapy for the treatment of meta‑ surgery (excision, debulking, or exenteration) (140). The early static UM. While BRAF inhibitors, such as dabrafenib and detection of radiation‑induced complications can allow their vemurafenib, have been used in cutaneous melanoma, which early treatment. Anti‑vascular endothelium growth factor typically harbors BRAF and NRAS mutations, there is no agents have been used for the treatment of radiation retinopathy, rationale for the use of these agents in UM due to the different radiation maculopathy, radiation‑induced optic neuropathy and molecular profile compared to cutaneous ones (153). Given neovascular glaucoma, and can help to stabilize or, in some the commonly harbored GNAQ/GNA11 mutations in UM, cases, to improve clinical conditions (141‑144). agents targeting downstream effectors of biological pathways As regards systemic surveillance, an ideal surveillance GNAQ/GNA11‑related, such as MEK and protein kinase C protocol which would define timing, duration and the type (PKC), have been investigated. However, similar to other ther‑ of examinations according to patient characteristics has not apeutic approaches, disappointing results have been reported yet been developed (62,106). Systemic monitoring is aimed and response rates, in general, are <10‑15% (114,147). MEK at the early detection of metastasis, which could have some inhibitors include selumetinib and trametinib. Initially, the use clinical relevance as highly selected cases of hepatic metas‑ of selumetinib in metastatic UM seemed to provide promising tasis could be managed with surgical resection, resulting in results: A randomized clinical trial enrolling 101 patients an improved survival (145). In addition, no adjuvant therapy compared selumetinib with traditional chemotherapy and has been demonstrated to be effective in reducing the risk reported longer progression‑free survival (PFS) and a higher of metastasis (146). In this scenario, risk stratification for response rate (14 vs. 0%) (154). However, these quite posi‑ metastasis development may play a key role and may allow for tive outcomes failed to be achieved in a subsequent phase III the planning of a surveillance protocol which could be suited randomized trial: The SUMIT trial compared selumetinib to individual risk (146,147). As aforementioned, prognosis is plus dacarbazine with dacarbazine alone in 129 patients with dependent on multiple factors, including clinical variables metastatic UM and revealed no significant difference in PFS and genetic prolife (GEP class). According to the GEP class, between the two interventions (median PFS, 2.8 months in patients can be classified as low‑risk (class I) and high‑risk the selumetinib plus dacarbazine group vs. 1.8 months in the (class II) (88). Surveillance imaging tends to be focused on dacarbazine alone group), and no difference in response rate hepatic monitoring due to the tendency of UM to metastasize (3% in selumetinib plus dacarbazine group vs. 0% in dacar‑ to the liver (146). Low‑risk patients can be monitored with bazine group) (155). Trametinib was investigated in a phase I hepatic ultrasound at 6‑month intervals (146). Hepatic ultra‑ trial enrolling 16 patients with metastatic UM and 81 patients sound features a good specificity (100%), but poor sensitivity with cutaneous or unknown primary melanoma. In patients (14%) (61). Therefore, high‑risk patients are recommended affected by UM, trametinib revealed no objective response to undergo a more frequent (<6‑month interval) and a more (0% response rate) and a median PFS of 1.8 months (156). intensive hepatic monitoring, which would include more sensi‑ Trametinib was also used in combination with Akt inhibitor: tive and specific imaging tests, such as liver CT/MRI (146). A randomized trial compared trametinib alone (18 patients However, other authors recommend annual liver ultrasound receiving ≥1 study drug dose) vs. trametinib combined with the and physical examination for low‑risk patients, and 6‑monthly Akt inhibitor GSK2141795 (21 patients receiving ≥1 study drug liver imaging (ultrasound alternated with liver/abdomen MRI) dose), revealing no difference in median PFS (15.7 weeks vs. plus annual physical examination for high‑risk patients (62). 15.6 weeks) and only one partial response in each group (157). Low‑risk and high‑risk patients can be transitioned to the GP Another biological pathway which has been studied as a at 5 and 10 years, respectively (62). possible target in UM is the MAPK pathway, through PKC 14 FALLICO et al: INSIGHTS INTO UVEAL MELANOMA inhibition. Sotrastaurin, a PKC inhibitor, has shown a median 19.2 months; there was no case of compete response, but 14 PFS of 15.4 weeks, a partial response in one patient and stable out of 24 patients had partial response or stable disease (167). disease in 55 patients (47%) (158). Furthermore, growth factor receptors which have been found overexpressed in UM, have 10. Genetic and epigenetic features in uveal melanoma been studied as possible targets. Sunitinib, a C‑kit inhibitor, was used in a pilot study on 20 patients with C‑kit expressing meta‑ Genetic and epigenetic characteristics of tumors have been static UM and demonstrawted a partial response in one patient given ever‑increasing attention over the past years, not only and 12 stable disease. The median PFS was 4.2 months (159). due to their relevant role in the carcinogenesis process, but also A retrospective study evaluated the use of sunitinib as an adju‑ as they can provide new insight into tumor behavior (168). This vant therapy in high‑risk UM and reported a longer overall can potentially allow the development of reliable biomarkers survival in those receiving adjuvant sunitinib compared to and novel therapeutic targets, leading to new breakthroughs in historical controls (160). Cabozantinib is a multiple tyrosine the management of UM. Genetic alterations affect directly and kinase receptor inhibitor, including c‑MET, Axl and VEGF. permanently the DNA sequence and include the following: This has been used in the treatment of metastatic UM. Very Chromosomal aberration, copy number variation (CNV), recently, the results of a RCT comparing cabozantinib vs. mutations either somatic or germline and single nucleotide dacarbazine or temozolomide, have been published and have polymorphism (SNP). Conversely, epigenetic alterations demonstrated no improvement in PFS in patients treated with modulate gene activity and expression without involving any cabozantinib (161). Sorafenib, multi‑kinase inhibitor, was used changes in the DNA sequence; these include the alteration of in a trial enrolling 152 patients with metastatic UM: Of the microRNA (miRNA/miR) expression levels, DNA methyla‑ 118 patients evaluable for response, 32.2% exhibited progres‑ tion and histone modifications (168). The interaction between sion, 1.7% had a partial response, and 66.1% had stable disease. genetic, epigenetic and other possible factors involved in UM Patients with stable disease were randomized to sorafenib carcinogenesis is illustrated in Fig. 1. continuation or placebo, with a median PFS significantly UM is a sporadic tumor, rarely exhibiting a familial longer in sorafenib arm (5.5 vs. 1.9 months) (162). inheritance (168). This is the case of tumor syndromes, such Since the liver represents the most common site for as Lynch syndrome (169) and BAP1‑tumor predisposition UM metastasis, several liver‑directed treatments have been syndrome (170). As reported above, chromosomal alterations investigated. A recent meta‑analysis of trials (phase Ib‑III) in UM mainly affect chromosomes 1,3,6 and 8, and present in metastatic UM revealed that liver directed treatments were a meaningful prognostic relevance. As regards somatic associated with a longer overall survival and PFS compared to mutations, the most commonly mutated genes that have other therapies (chemotherapy, immunotherapy and targeted been identified in UM patients are GNAQ, GNA11 mainly therapy) (163). Liver directed treatments include surgical affected by specific point mutations (p.Q209P and p.Q209L, resection, stereotactic radiotherapy, radiofrequency ablation, respectively) and BAP1 that is subjected to several mutations regional chemotherapy and embolization (114,164). Surgical occurring in the whole gene sequence (88,171). Unlike cuta‑ resection could be a therapeutic option in very selected neous melanoma, UM does not harbor typical mutations in cases. A study including 155 patients with liver metastases BRAF and NRAS genes (153,172). The GNAQ and GNA11 from UM reported that 11% of patients underwent liver genes encode for the Gα11 subunits and the Gαq subunits of G resection, with a better survival compared with those who proteins, respectively (171). G proteins are involved in signal did not receive surgical resection (145). Regional chemo‑ transduction, controlling gene transcription and, subsequently, therapy includes hepatic intra‑arterial infusion and isolated cell survival, growth and mortality (171). GNAQ and GNA11 hepatic perfusion (IHP). Fotemustine has been shown to are located on chromosome 9 (q21.2) and chromosome 19 have a better response rate (10.5 vs. 2.5%) and longer PFS (p13.3), respectively (168). These genes have been considered (4.5 vs. 3.5 months) when administered as intra‑arterial perfu‑ as driver oncogenes, representing early or initiating mutations sion compared as intravenously, but with no difference in in UM (168,171). Oncogenic mutations of GNAQ and GNA11 overall survival (165). Isolated hepatic perfusion is based on genes are usually mutually exclusive and were found in up to the isolation of liver blood supply from systemic circulation 83% of Ums (173). Oncogenic mutations of these genes deter‑ with the purpose of delivering high‑dose chemotherapy and mine a constitutive activation of G proteins, which, in turn, reducing at the same time systemic exposure. A retrospec‑ affect downstream signaling (171). Several intracellular path‑ tive study on 18 patients treated with percutaneous IHP with ways are regulated by GNAQ and GNA11 genes, including the melphalan reported a median overall survival of 9.6 months RAF/mitogen‑activated protein kinase kinase (MEK)/extra‑ and a median PFS of 12.4 months (166). Embolization cellular signal‑regulated kinase (ERK) pathway (174). The treatment modalities include chemoembolization, radioembo‑ activation of the RAF/MEK/ERK pathway causes an overex‑ lization and immunoembolization (164). A recent prospective pression of cell‑cycle regulatory protein cyclin D1 (CCND1), trial investigated radioembolizaton in two groups of patients: leading to the inactivation of the tumor Treatment‑naïve patients and patients who exhibited progres‑ suppressor gene (174). Mutations in this pathway have been sion following immunoembolization. In the treatment‑naïve assumed to be early or initiating events in UM carcinogen‑ group, median PFS was 8.1 months and median overall survival esis (174). The BAP1 gene is an onco‑suppressor gene located 18.5 months; no case of complete response was found, but 20 on chromosome 3 (p21.1). Inactivating somatic mutations of out of 23 patients had a partial response or stable disease. BAP1 have been associated with metastatic disease: Up to In the group resistant to immunoembolization, the median 84% of metastasizing UMs harbor inactivating mutations of PFS was 5.2 months, and the median overall survival was this onco‑suppressor gene (32). As regards BAP1 function, this INTERNATIONAL JOURNAL OF ONCOLOGY 58: 10, 2021 15

Figure 1. Schematic representation of the genetic and epigenetic alterations and risk factors involved in the development of uveal melanoma. Environmental and individual risk factors (including age, sex and ethnicity) are able to induce both genetic and epigenetic modifications responsible for the malignant transformation of choroid cells. Clinical and prognostic assessment could benefit from the analysis of genetic and epigenetic factors associated with the development of uveal melanoma. is a deubiquitinating enzyme that regulates specific proteins. hypomethylation and hypermethylation of widespread regions In particular, BAP1 regulates genes involved in of the tumor cell genome (180). function and differentiation: Inactivating mutations can lead Identifying the gene regions affected by methylation to melanocytic de‑differentiation, promoting a pro‑metastatic phenomena is important to establish the effect that this modi‑ behavior. The loss of the tumor suppressor activity of BAP1 fication determines at the transcriptional level. It is currently has been identified in breast and lung cancers as well (175). Of known that promoter methylation determines gene suppres‑ note, BAP1 mutations can also occur in germline featuring the sion by blocking the access of transcription factors to binding BAP1 familial cancer syndrome, which predisposes to several sites on the promoter (181,182), while it has not yet been cancers, including UM (176). fully clarified the functional role of intragenic and intergenic As regards epigenetic alterations, biological mechanisms methylation in the control of gene expression (183). On this related to miRNAs and DNA methylation have been identified matter, several studies have described how DNA methylation in UM, whereas modest evidence on histone modification is can modulate the expression of non‑coding RNA, the alterna‑ available. The role of histone modification has been associ‑ tive splicing, the recruitment of enhancers, and the increase ated with BAP1 loss. It has been shown that BAP1 depletion of RNA polymerase activity that influences the expression determines a loss of differentiation in cancer cells through the levels of the genes involved (184‑186). As regards UM, the hyperubiquitination of histone H2A (175). The use of histone hypomethylation of sites close to the preferentially expressed deacetylases inhibitors proved to reverse H2A hyperubiqui‑ antigen in melanoma (PRAME) promoter has been shown to tination, inducing cell differentiation and inhibiting tumor promote PRAME activation with subsequent increase in meta‑ growth (175). Indeed, the nuclear expression of several histone static risk (187). The hypomethylation of the deleted in split deacetylases has been found in UMs, confirming the role of hand/split foot 1 (DSS1) promoter has been found to be a this epigenetic alteration as a potential therapeutic target (177). frequent event in UM (188). Hypermethylation of the following DNA methylation represents an important epigenetic oncosuppressor gene promoters has been demonstrated in mechanism which regulates the expression of several genes UM: p16, RASSF1A, RASEF, TIMP3 and EFS (189‑194). This involved in UM carcinogenesis. This regulation is based on event leads to the inactivation of these genes. The methylation methylation/demethylation mechanisms, that is the addi‑ of human telomerase reverse transcriptase (TERT) promoter tion/removal of a methyl group (CH3) to/from a DNA sequence has been described in UM. Human TERT is an oncogene mediated by DNMT3A and TET1. It has been widely demon‑ which has been found to be upregulated in UM (190). TRAIL strated that certain methylated DNA regions can be used receptors DcR1 and DcR2 were found hypermethylated in both as stable biomarkers, considering that DNA methylation is UMs and cutaneous melanomas (195). Of note, the hypermeth‑ maintained almost unchanged during the cell replication ylation of a site on chromosome 3 at BAP1 locus determines process by DNA methyltransferase 1 (DNMT1) (178,179). The BAP1 downregulation, showing the epigenetic regulation of functional alteration of this enzyme, together with that of other this gene (196). methyltransferases (DNMT3A and DNMT3B) that catalyze miRNA‑based epigenetic mechanisms have been also the de novo DNA methylation, is responsible for the global investigated in UM (197). miRNAs are non‑coding RNA 16 FALLICO et al: INSIGHTS INTO UVEAL MELANOMA molecules, consisting of a single‑stranded sequence of generation sequencing and droplet digital PCR (ddPCR), have 18‑22 nucleotides. miRNAs regulate gene expression, playing been used for the analysis of liquid biopsy samples to detect important roles in both physiological and pathological low amounts of miRNAs, circulating mutations, microbial processes, such as cell proliferation, differentiation, apoptosis, nucleic acids, etc. (227‑229). organ formation, angiogenesis, extracellular matrix remodeling, Therefore, the analysis of both genetic and epigenetic etc. (198,199). The dysregulation of specific miRNAs has been factors in liquid biopsy and tissue samples obtained from associated with the onset and progression of a number of types patients with UM by using these high‑sensitive techniques of cancer, including UM (200‑203). Research in this field has would contribute to the discovery of novel effective diagnostic exhibited marked progress. Venza et al found 96 miRNAs and prognostic biomarkers for the management of UM. dysregulated in both UM and cutaneous melanoma cell lines (204). Radhakrishnan et al found specific miRNAs 11. Conclusions associated with metastatic UM, which were in association with chromosome 1,3 and 8 aberrations (205). However, Larsen et al The present review article focused on UM and collected an failed to demonstrate the role of this association between updated evidence on epidemiological, clinical and molecular chromosome alterations and miRNAs expression in predicting aspects of this malignancy. It is suggested that even if UM metastatic disease in UM (206). Worley et al demonstrated that is a relatively rare type of cancer, the prognosis remains the expression of 6 miRNAs (let‑7b, miR‑199a*, miR‑199a, unfavorable in a consistent percentage of cases. The treatment miR‑193b, miR‑143 and miR‑652) could be used to distinguish of the primary tumor has made significant improvements class1 UM (low metastatic risk) from class 2 ones (high metastatic thanks to the introduction of globe‑preserving approaches in risk) with maximum sensitivity and specificity (207). Among the previous century. However, metastatic disease remains a the miRNAs involved in cell proliferation and apoptosis, which critical issue for clinicians due to the lack of effective thera‑ have been found abnormally expressed in UM, noteworthy peutic strategies. Molecular studies have provided new insight are the following: miR‑137 (downregulated); miR‑144 into the genetic and epigenetic mechanisms that regulate (downregulated); miR‑145 (downregulated); miR‑92a‑3p UM biological activity. However, it seems that the available (upregulated); and miR‑181b (upregulated) (208‑213). Among knowledge has only scratched the surface and there is hope the miRNAs involved in cell migration and invasion, which for the identification of effective biomarkers and new therapy have been found abnormally expressed in UM, noteworthy are which can make the difference in the management of meta‑ the following: miR‑20a (upregulated); miR‑155 (upregulated); static disease. This could be the breakthrough of the current miR‑296‑3p (downregulated); miR‑454 (upregulated); miR‑367 century. (upregulated); miR‑21 (upregulated); miR‑23a (downregulated); and miR‑224‑5p (downregulated) (214‑221). Additionally, Acknowledgements miR‑204 and miR‑145 have been found to be downregulated, whereas miR‑20a, miR‑17, miR‑106a, miR‑34a and miR‑21 Not applicable. have been found to be upregulated in UM samples (222). Circulating levels of the following miRNAs have been found Funding to be dysregulated in metastatic UM: miR‑125b, miR‑20a, miR‑146a, miR‑181a, miR‑155 and miR‑223 (223). miRNAs The present study was partially supported by the Italian are also involved in the regulation of immune mediators, which League Against Cancer (LILT). can modulate UM behavior. For instance, interleukin (IL)‑10 may play a role in promoting cancer as can suppress immune Availability of data and materials response against the tumor. Specific miRNAs seem to be involved in IL‑10 modulation (224). miRNAs may also play Not applicable. a promising role in the development of therapeutic targets. For instance, genistein, an antitumor drug, has been shown to Authors' contributions inhibit miR‑27a expression and, as a result, to increase ZBTB10 gene expression (miR‑27a target gene); the antitumor action MF, LF and TA conceived the study. MF and GR wrote the may be related to the miR‑27a regulatory mechanism (225). original draft of the manuscript. AL, MR, VB, AR, GG and Overall, all these studies provide important insight into the LF performed the bibliography research and collected the involvement of miRNAs in UM development and progression, data. RC collected data on the pathological staging of uveal as well as in the prognosis of UM patient. However, some data melanoma. MF, GG and LF prepared the figure and tables. are still conflicting; therefore, there is an urgent need to clarify All authors contributed to the critical revisions, editing and which miRNAs are effectively involved in UM development or reviewing of the final version of the manuscript. in specific clinical‑pathological features of patients. Recent studies have proposed the evaluation of all these Ethics approval and consent to participate genetic and epigenetic biomarkers for the early diagnosis of tumors. In particular, a previous study demonstrated how the Not applicable. analysis of liquid biopsy samples and circulating DNA may be useful for the early detection of genetic aberrations and epigen‑ Patient consent for publication etic markers of precancerous and cancerous lesions (226). In this context, novel high‑sensitive techniques, such as next Not applicable. INTERNATIONAL JOURNAL OF ONCOLOGY 58: 10, 2021 17

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